| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Biology, Division of Botany & Plant Physiology, University of Oslo, P.O. Box 1045, Blindern, N-0316 Oslo, Norway
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
The population structure of 11 Fennoscandian geographic populations of the pioneer wood-decay basidiomycete Trichaptum abietinum was assessed with PCR-RFLPs, intersequence simple repeats (ISSRs) and mating studies. The three codominant PCR-RFLP markers (1) internal transcribed spacer 2 (nrDNA), (2) glyceraldehyde-3-phosphate dehydrogenase and (3) translation elongation factor 1
showed that genotype distributions in most cases (94%) agreed with Hardy-Weinberg expectations and that random association of alleles occurred across loci. The molecular data suggest that T. abietinum is a highly outcrossing fungus that regularly proliferates and spreads by sexual spores. Interstock mating reactions suggest a high number of mating factors among individuals and that biological barriers to gene flow are nonexistent in the region. The three PCR-RFLP loci gave an overall FST = 0.03, indicating a low level of genetic differentiation and presumably high gene flow among the geographic populations. The ISSR markers revealed no systematic substructuring and the among-population variance component was low (6.1%) in AMOVA. However, all PCR-RFLP and most ISSR markers (7/12) showed significant deviation from the null hypothesis of an even distribution of allele frequencies across the 11 geographic populations. Allele frequencies varied in an apparently random manner, suggesting that genetic drift might be an important structuring factor in T. abietinum. The spatial small-scale distribution of heterokaryons on three selected substrate units (logs) showed that most isolates represented discrete individuals and that a number of genets (19) may occupy a single log. The small-scale genotype distributions (within logs) were in agreement with panmictic Hardy-Weinberg expectations.
Key words: Basidiomycota, genetic equilibrium, ISSR, PCR-RFLP
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
).
The population structure and life history traits of polypores are largely unexplored. The fact that most polypores have wide geographic distributions (Ryvarden and Gilbertson 1993) might reflect good ability for dispersal. Little genetic differentiation was observed among northern European populations of Fomitopsis pinicola (Swartz : Fr.) Karst. (Nordén 1997, Högberg et al 1999), indicating effective dispersal capacity and large population sizes. In contrast, significant genetic differentiation was detected among European populations of Heterobasidion annosum (Fr.) Bref. (Stenlid et al 1994) and Fomitopsis rosea (Alb. et Schw. : Fr.) P. Karst. (Högberg and Stenlid 1999). In other wood-degrading basidiomycetes, e.g., Schizophyllum commune Fr., Phlebiopsis gigantea (Fr.) Jül. and Pleurotus tuberregium (Fr.) Sing., a significant genetic differentiation between continents likewise was observed, indicating limited gene flow at this spatial scale (Isikhuemhen et al 2000, Vainio and Hantula 2000, James et al 2001).
Fungi exhibit highly variable life-history strategies, which are believed to have great impact on their population structure. T. abietinum expectedly has, like most basidiomycetes, a predominant heterokaryotic (dikaryotic) vegetative stage and a transitory homokaryotic (monokaryotic) stage after meiosis and before heterokaryon formation. Clonal dispersal, which is common in Ascomycota, seems less prevalent in basidiomycetes but has been reported in some species, e.g., in Amylostereum areolatum (Fr.) Boid. and A. chailletii (Pets. : Fr.) Boid. (Vasiliauskas et al 1998). On the other hand, intersterility barriers causing limited gene flow and accelerated genetic differentiation commonly are observed in polypores and in the long-term might lead to sympatric or parapatric speciation. In T. abietinum, two intersterility groups have been found in North America and one group in Europe. While the North American groups apparently are intersterile, the European group is interfertile with both the North American groups (Macrae 1967, Magasi 1976). The mating system also might affect the fungus population structure. It is well documented that T. abietinum has a heterothallic (outcrossing) tetrapolar mating system (Macrae 1967, Magasi 1976). Compared with a bipolar mating system exhibited in most polypores investigated to date, the tetrapolar mating system significantly minimizes the possibility for inbreeding.
Our aim was to investigate the population structure and genetic diversity in T. abietinum on a regional and local spatial scale. Eleven geographic populations of T. abietinum in Norway, Sweden and Finland were included. Ten populations were located in the continuous belt of the northern boreal coniferous forest and one population in western Norway, isolated from the other populations by high mountains. The sampling strategy was chosen to assess whether the geographic populations belonged to a single mating population or represented several discrete populations. We employed mating compatibility analyses to study the putative occurrence of intersterility barriers across geographic populations, and locus-specific codominant PCR-RFLP and anonymous intersequence simple-repeat (ISSR) markers to investigate the population structure and whether substructuring exists among geographic populations. We also performed a small-scale study of the spatial distribution and possibly nonrandom mating of individuals on three selected substrate units (logs) in one population.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
|
Molecular methods –
DNA was extracted from all isolates by the CTAB miniprep method described by Murray and Thompson (1980) with some minor modifications: DNA was re-suspended in 100 µL sterile filtered H2O at the final step of extraction; and DNA templates were diluted 50-fold before PCR-amplification. Amplification was accomplished with primers ITS3 and ITS4 (White et al 1990) for the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA (nrDNA), the primers CTK-052 (5'-CGGCCGTATCGTCCTCCGTAATGC) and CTK-032 (5'-GAGTAACCGCATTCGTTATCGTACC) for the glyceraldehyde-3-phosphate dehydrogenase (gpd) region (Kreuzinger et al 1996), the primers TaGPD2F (5'-ACACCGGTCGATTCGACAATG) and CTK-052 for the partial region of gpd, and the primers EF595F (5'-CGTGACTTCATCAAGAACATG) and EF1160R (5'-CCGATCTTGTAGACGTCCTG) (Kauserud and Schumacher 2001) for the partial translation elongation factor (efa). The Ta-GPD2F primer was designed to fit within the gpd sequences. PCR was performed in 40 µL reactions containing 19.5 µL 50x diluted template and 20.5 µL reaction mix (final concentrations: 250 µmol/L dNTPs), 0.625 µmol/L of each primer, 2 mmol/L MgCl2 and 1 unit DyNazymeTM II DNA polymerase [Finnzymes Oy, Espoo, Finland] on a Genius Operator (Techne) or Biometra PCR machine. The ITS2 amplification started with a denaturation step for 4 min at 94 C, followed by 36 cycles of 30 s, denaturation at 94 C, 35 s annealing at 52 C, extension at 72 C for 40 s, and a final extension step at 72 C for 10 min before storage (4 C). A similar thermal profile was used in the amplification of the partial efa and gpd sequences, except that the annealing was optimized to 55 C (partial efa and gpd) and 54 C (partial gpd).
ISSR amplification was performed in 40 µL reactions in the same reaction mixture and concentrations as in the PCR reactions, with the exception that the concentration of the single ISSR primer (GGGC[GA]8) (Becker and Heun 1995) was doubled. PCR conditions for ISSR reactions were: 5 min denaturing at 95 C; one cycle of 30 s at 95 C, 2.5 min at 92 C, annealing 1 min at 55 C, and 2 min at 72 C; and 44 cycles of 1 min at 92 C, 1 min at 55 C, and 2 min at 72 C and 10 min extension at 72 C (Becker and Heun 1995). Independent reiterated PCR amplifications were performed from a subset of isolates (16) to ensure the reproducibility of the ISSR markers.
PCR products of homokaryons were sequenced manually with PCR primers as sequencing primers, employing the ThermoSequenase radiolabeled terminator cycle sequencing kit (Amersham Pharmacia Biotech Inc., OH, USA) and -33P-ddNTPs. All sequences are deposited in the EMBL nucleotide database with accession numbers AJ309814–309815 (ITS2), AJ309882–309891 (partial efa), and AJ309892–AJ309901 (partial gpd). In conferring with the endonuclease database Webcutter 2.0, we noticed that the endonucleases RsaI, HhaI and DdeI would give polymorphism in the ITS2, efa and gpd sequences. The three PCR-RFLP markers were situated in noncoding spacer sequences. For restriction analyses, 10 µL of ITS2, efa and gpd amplicons were digested in 25 µL volumes containg 16.5 µL H2O, 2.5 µL buffer and 0.5 µL enzyme, following the manufacturer's instructions (Promega). ISSR and restriction products were separated on 2% agarose gels and stained with ethidium bromide, using 0.5 TBE as running buffers. Results were recorded by photographing the gels over UV light.
Statistical analyses –
The PCR-RFLP and ISSR dataset included 223 heterokaryotic isolates sorted according to geographical origin, in addition to 57 isolates from the small-scale study. The biallelic PCR-RFLP loci were scored on presence or absence of restriction sites. The ISSR bands were scored as present (1) or absent (0) across all populations. Chi square tests for homogeneity of allele frequency distributions across the geographic populations (by constructing two-way contingency tables), observed and expected heterozygosity, and Wright's fixation index (FIS) were calculated in POPGENE version 1.32 (Yeh et al 1997). Linkage disequilibrium between pairs of PCR-RFLP markers was tested in the program Arlequin version 2.0 (Schneider et al 2000) using the approach given by Slatkin and Excoffier (1996) with 1000 permutations and 10 initial conditions. Any deviation from Hardy-Weinberg equilibrium in the 11 geographic populations was assessed for the PCR-RFLP markers by the exact probability test (Guo and Thompson 1992), implemented in Arlequin ver. 2.0, with 1000 steps in Markov chain and 1000 dememorization steps. Analysis of molecular variance (AMOVA), calculation of overall FST and significance test of FST also were performed in Arlequin ver. 2.0 with the approach given by Weir and Cockerham (1984) with 1000 permutations. Unweighted Pair Group Method with Arithmetic Averaging (UPGMA) and Principal Component Analysis (PCO) of the ISSR data was performed in NTSYSpc2.02 (Rohlf 1994).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
|
|
2 tests, p < 0.05). The PCR-RFLP allele frequencies varied in an apparently random manner across populations (Fig. 3).
|
2 tests, p < 0.05) from the null hypothesis of a homogenous distribution (Table II).
|
|
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
). In light of the outcrossing reproductive mode and the high abundance of T. abietinum and F. pinicola in this region, it is not surprising that the population genetic analyses suggest panmictic conditions in these species. The high frequency (95%) of compatible interstock pairings among thirteen homokaryons from a single place indicates a multiallelic breeding system. The mating loci of basidiomycetes are believed to display strongly balanced polymorphism (May 1999), resulting in higher fitness for infrequent mating alleles than for frequent alleles. Such selection is expected to counter genetic drift and, within a mating population, the maintenance of a high number of mating alleles is expected, as was observed in T. abietinum. As far as we know, a multiallelic breeding system has been observed in all basidiomycetes investigated.
Inbreeding coefficient statistics gave a low overall FST (0.03), which indicates little genetic differentiation across populations in T. abietinum. Little genetic differentiation also was observed in northern European populations of F. pinicola (FST = 0) (Högberg et al 1999) and F. rosea (FST = -0.02) (Högberg and Stenlid 1999). In T. abietinum, ISSR data provided no discrete clustering of the various geographic populations in the PCO analysis, and little among-populations variance (6.1%) was recorded in AMOVA. Thus, no distinct genetic substructuring was recorded, and the data suggest large and widespread populations with high migration rates. Trichaptum abietinum is an early colonizer of dead coniferous wood, a short-lived habitat that might be considered as "sinking islands" that must be colonized, exploited and abandoned in short time, we estimate roughly 1–3 years for T. abietinum. These conditions obviously require expansive disperal abilities. Likewise, very little genetic divergence occurred among northern European populations of the early colonizers Cylindrobasidium evolvens (Fr.) Jülich (Vasiliauskas and Stenlid 1998), Amylostereum areolatum and A. chailletii (Vasiliauskas et al 1998, Vasiliauskas and Stenlid 1999), by employing random amplified DNA markers (AP-PCR), and in P. gigantea, employing RAMS markers (Vainio and Hantula 2000). Overall, very little genetic substructuring among wood-inhabiting basidiomycetes has been observed on an intracontinental scale.
However, tests for homogeneity of allele frequencies across geographic populations showed significant divergence from the null hypothesis of a homogeneous distribution in all three PCR-RFLP markers and seven out of 12 ISSR markers. The variation was not distributed according to geographic origin and is difficult to interpret. One possible explanation might be that the stochastic process of genetic drift is highly involved in the structuring of T. abietinum populations. This might imply that the fungus experiences a high population turnover and rapid shifts in local population sizes, which are factors that increase the influence of genetic drift. Genetic drift through founder events has been suggested as an important moderating evolutionary factor in plant pathogenic fungi (Carlier et al 1996) and might be influential on early colonizers of dead wood, as well.
The UPGMA analysis of ISSR data showed that the Voss population was genetically divergent from the other populations. A private gpd restriction site (allele) also appeared in two individuals from this population. Isolated populations, such as the Voss population in a minor spruce forest in western Norway delimited from the other geographic populations by high mountains, are more exposed to genetic drift. A founder event might have occurred during the establishment of the Voss population. Furthermore, heterozygote deficits were observed in all three loci in the Voss population, a finding that might be explained by a limited population size and inbreeding.
Intraspecific barriers to gene flow are a structuring factor believed to be important in some polypores. This might lead to the development of different intersterility groups (ISGs). In Heterobasidion annosum, several genetically differentiated intersterility groups are recognized (Stenlid et al 1994, Garbelotto et al 1996). In T. abietinum, we obtained compatible matings (98.8%) between homokaryons from a subset of the geographic populations, suggesting that the actual homokaryons belonged to the same intersterility group. Two intersterile North American groups and one European group, partially fertile with the North American groups, have been inferred from T. abietinum (Macrae 1967, Magasi 1976). Our results support the conclusion that only one intersterility group of T. abietinum occurs in Fennoscandia. The slightly higher portion of compatible interstock matings between the geographic populations (98.8%), compared to matings within the Skotjernfjell population (94.9%), might be due to a higher proportion of shared mating factors in the latter population.
In the small-scale analysis, most isolates represented discrete individuals and as many as 19 genets occupied a single log. The occurrence of multiple colonies of polypores on single substrate units has been demonstrated, e.g., in Phellinus tremulae (Bondartzev) Bondartzev & Borissov (Holmer et al 1994), F. pinicola (Nordén 1997), and H. annosum (Garbelotto et al 1999). In T. abietinum, we observed four groups of isolates with identical multilocus ISSR/PCR-RFLP genotypes, which probably represent four different genets that produced fruit bodies on the logs. However, most isolates (91.2%) possessed a unique multilocus profile, which suggests that genets of T. abietinum share small parts of the substrate, a feature that is assumed to be typical for many early colonizers. The genotype distributions on the logs fulfilled Hardy-Weinberg expectations, suggesting that random mating also was predominant at this scale (single log). Average heterozygote deficits were observed on all three logs, and linkage disequilibria between some loci might indicate some nonrandom mating or local dispersal. He-ho mating was evidenced in vitro and might prove to be an important phenomenon in shaping the small-scale population structure of wood-inhabiting basidiomycetes in nature. In a study of P. ostreatus, a nonrandom distribution of mating factors was observed at the level of a single substrate, suggesting mating between relatives (Kay and Vilgalys 1992). Neighboring isolates also were shown to share the same mating factor and in some cases apparently the same nuclei in H. annosum, suggesting that he-ho mating has taken place in co-occurring field isolates (Garbelotto et al 1999). However, the nonrandom distribution of mating factors might have been attributed to local dispersal of spores from the same fruit body.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Accepted for publication October 9, 2002.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Carlier J, Lebrun H, Zapater MF, Dubois C, Mourichon X., 1996 Genetic structure of the global population of banana black leaf streak fungus, Mycosphaerella fijiensis. Mol Ecol 5:499-510
Garbelotto M, Ratcliff A, Bruns T, Cobb FW, Otrosina WJ., 1996 Use of taxon-specific competitive-priming PCR to study host specificity, hybridization, and intergroup gene flow in intersterility groups of Heterobasidion annosum. Phytopathology 86:543-551
———, Cobb FW, Bruns TD, Otrosina WJ, Popenuck T, Slaughter G., 1999 Genetic structure of Heterobasidion annosum in white fir mortality centers in California. Phytopathology 89:546-554
Guo S, Thompson E., 1992 Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics 48:361-372[Medline]
Hantula J, Dusabenyagasani M, Hamelin RC., 1996 Random amplified microsatellites (RAMS)—a novel method for characterizing genetic variation within fungi. European Journal of Forest Pathology 26:159-166
Holmer L, Nitare L, Stenlid J., 1994 Populations structure and decay pattern of Phellinus tremula in Populus tremula as determined by somatic incompatibility. Can J Bot 72:1391-1396
Högberg N, Holdenreider O, Stenlid J., 1999 Population structure of the wood decay fungus Fomitopsis pinicola. Heredity 83:354-360
———, Stenlid J., 1999 Population genetics of Fomitopsis rosea—a wood-decay fungus of the old-growth European taiga. Mol Ecol 8:703-710
Isikhuemhen OS, Moncalvo JM, Nerud F, Vilgalys R., 2000 Mating compatibility and phylogeography in Pleurotus tuberregium. Mycol Res 104:732-737
James TY, Moncalvo JM, Li S, Vilgalys R., 2001 Polymorphism and the ribosomal DNA spacers and its relation to breeding structure of the widespread mushroom Schizophyllum commune. Genetics 157:149-161
Kauserud H, Schumacher T., 2001 Outcrossing or inbreeding: DNA markers provide evidence for type of reproductive mode in Phellinus nigrolimtatus (Basidiomycota). Mycol Res 105:676-683
Kay E, Vilgalys R., 1992 Spatial distribution and genetic relationship among individuals in a natural population of the oyster mushroom Pleurotus ostreatus. Mycologia 84:173-182
Kreuzinger N, Podeu R, Gruber F, Göbl F, Kubicek CP., 1996 Identification of some ectomycorrhizal basidiomycetes by PCR amplification of their gpd (Glyceraldehyde-3-Phosphate Dehydrogenase) genes. Appl Environ Microbiol 62:3432-3438[Abstract]
Macrae R., 1967 Pairing incompatibility and other distinctions among Hirschioporus [Polyporus] abietinus, H. fusco-violaceus, and H. laricinus. Can J Bot 45:1371-1398
Magasi LP., 1976 Incompatibility factors in Polyporus abietinus, their numbers and distribution. Memoirs of the New York Botanical Garden 28:163-173
May G, Shaw F, Badrane H, Vekemans X., 1999 The signature of balancing selection: fungal mating compatibility gene evolution. Proc Natl Acad Sci USA 96:9172-9177
Murray MG, Thompson WF., 1980 Rapid isolation of high molecular weight plant DNA. Nucl Acid Res 8:4321-4325
Nordén B., 1997 Genetic variation within and among populations of Fomitopsis pinicola (Basidiomycetes). Nord J Bot 17:319-329
Olofsson D., 1996 Tickor i Sverige. Projektrapport 1996
Rohlf FJ., 1994 NTSYSpc. Numerical taxonomy and multivariate analysis system, version 2.02. Exceter Software, New York, USA
Ryvarden L, Gilbertson RL., 1994 European polypores. Fungiflora, Oslo, Norway
Schneider S, Roessli D, Excoffier L., 2000 ARLEQUIN ver 2.0. A software for population genetics data analysis. Geneva, Switzerland
Slatkin M, Excoffier L., 1996 Testing for linkage disequilibrium in genotypic data using the EM algorithm. Heredity 76:377-383
Stenlid J, Karlsson JO, Högberg N., 1994 Intraspecific genetic variation in Heterobasidion annosum revealed by amplification of minisatellite DNA. Mycol Res 98:57-63
Vainio EJ, Hantula J., 2000 Genetic differentiation between European and North American populations of Phlebiopsis gigantea. Mycologia 92:436-446
Vasiliauskas R, Stenlid J., 1998 Population structure and genetic variation in Cylindrobasidium evolvens. Mycol Res 102:1453-1458
———, ———. 1999 Vegetative compatibility groups of Amylostereum areolatum and A. chailletii from Sweden and Lithuania. Mycol Res 103:824-829
———, ———, Thomsen IM., 1998 Clonality and genetic variation in Amylostereum areolatum and A. chailletii from Northern Europe. New Phytol 139:751-758
Weir BS, Cockerham CC., 1984 Estimating F-statistics for the analysis of population structure. Evolution 38:1358-1370
White TJ, Bruns T, Lee S, Taylor J., 1990 Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods and applications. San Diego,California: Academic Press
Yeh FC, Yang RC, Boyle TB, Ye ZH, Mao JX., 1997 POPGENE, the user-friendly shareware for population genetic analysis. Molecular Biology and Biotechnology Centre, University of Alberta, Canada
This article has been cited by other articles:
 |
|
 |
|
  P. M. Letcher, M. J. Powell, J. G. Chambers, and W. E. Holznagel Phylogenetic relationships among Rhizophydium isolates from North America and Australia Mycologia, November 1, 2004; 96(6): 1339 - 1351. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
